Insect antimicrobial peptide is a class of small-molecule alkaline polypeptide, which has heat stability, “non-specific inducibility and a broad-bactericidal spectrum”. It is promising to develop a new type of polypeptide antibiotic with insect antimicrobial peptide. It is known that about 15-20 kinds of bactericidal proteins, which include lysozyme, attacin, antibacterial peptide and other three classes, are produced in the haemolymph of virus infected Hyolophora cecropia (Hultmark D. et al, Eur. J. Biochem. 106: 7-16, 1980). According to small differences of the primary structure, Hyolophora cecropia is classified under three types: A, B and D (Hultmark D, same as above). Besides, a series of similar antimicrobial peptides have been separated from Chinese Antheraea pernyi. These polypeptides, which are obtained from various insects and can kill plant pathogens, are collectively referred to as antimicrobial peptides. Their amino acid sequences have high homology with each other, and there are large numbers of hydrophilic amino acid residues, especially lysine, arginine and alkaline amino acids in these molecules, while there are much more hydrophobic residues at C-end. Moreover, there are lots of conserved amino acid residues at many specific positions within the polypeptides, such as tryptophan in position 2, lysine in position 5, 8 and 9, aspartate in position 11 and 20 and so on (Steiner H. et al, Nature 292: 246-248, 1981).
In the world, many researches on antimicrobial peptide genes engineering have been carried out because antimicrobial peptides have a large potential as sterilizing or anti-tumor agent and in inhibiting virus copies. Up to now, expression researches on antimicrobial peptide genes in the Colibacillus, yeasts and baculovirus carriers have been reported.
“Study on expression of antibacterial peptide AD genes in AcNPV vector expression system” (Huang Ya-dong, Chinese Journal of Antibiotics, 28:304-307, 2003) reported a method of studying antibacterial peptide AD (CAD) genes expression “in insect culture cell” and insect by baculovirus vector system. CAD genes were modified by polymerase chain reaction (PCR) amplification. The modified CAD genes were first cloned into plasmid pGEM-T easy vector for identification and sequence analysis, and then subcloned into Autographa californica nuclear polyhedrosis virus (AcNPV) vector pAcGP67B to obtain a recombinant virus vector. Spodoptera frugiperododa Sf9 cells were co-infected with a recombinant virus vector and wild type AcNPVDNA, and recombinant viruses were screened through Sea Plaque screening. Sf9 cell and Autographa californica young larvae were transfected by the recombinant virus AcNPVAD, the CAD genes which were used for antibacterial activity test have been confirmed to be expressed in baculovirus carriers system, and the expression products has antibacterial activity.
The publication “Modification of antibacterial peptide AD genes and its expression in Pichia pastoris” (Huang Ya-dong, Journal of South China University of Technology (Natural Science Edition) 30: 13-16, 2003) reported that antibacterial peptide AD genes were amplified and modified with Asn codon added to its C terminal end by PCR. The modified antibacterial peptide AD genes were cloned into the integrative plasmid pPICZ-A to construct a recombinant expression vector, and then transformed into Pichia pastoris host bacterium GS115. The recombinant transformants were screened by using a selective agar media that contains a zeocin resistant mark. After fermentation, the concentrate was analyzed and tested on acidic-PAGE for its antibacterial activity. The results showed that antibacterial peptide AD genes was successfully expressed in Pichia pastoris. The expression product was secreted outside with the guidance of a factor signal and had a strong antibacterial activity.
The patent ZL96100376.6 disclosed four kinds of polypeptides or derivatives with antibacterial activity and antimicrobial peptide production methods thereof in cultured cells such as Colibacillus cells, yeast cells, plant cells, insect cells, mammalian cells and so on.
All of the above mentioned methods use cultured virus, Colibacillus cells, yeast cells, plant cells, insect cells, mammalian cells and so on for expressing genes coding antimicrobial peptides. But there are some disadvantages using virus vector system to express the objective proteins like:                cumbersome experimental procedures,        difficulties to control the experimental conditions,        not suitable for mass production.        
Antimicrobial peptide had inhibiting effect on bacteria such as E. coli. So E. coli, as a prokaryote, was difficult to use as an expression system. Although antimicrobial peptide had no inhibiting effect on eukaryotic cells, studies showed that it was difficult to achieve high-density cultivation, and had a greater impact on improving the expression level in yeast. Moreover, its amidation in yeast was incomplete, which had some impacts on its sterilizing activity. Moreover, the eukaryotic cells expression system had the disadvantage of long production cycles and high production costs.